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Curran, Samuel C., Pereira, Jose H., Baluyot, Marian-Joy, Lake, Julie, Puetz, Hendrik, Rosenburg, Daniel J., Adams, Paul and Keasling, Jay D. (2020). Structure and Function of BorB, the Type II Thioesterase from the Borrelidin Biosynthetic Gene Cluster. Biochemistry, 59 (16). S. 1630 - 1640. WASHINGTON: AMER CHEMICAL SOC. ISSN 0006-2960

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Abstract

alpha/beta hydrolases make up a large and diverse protein superfamily. In natural product biosynthesis, cis-acting thioesterase alpha/beta hydrolases can terminate biosynthetic assembly lines and release products by hydrolyzing or cyclizing the biosynthetic intermediate. Thioesterases can also act in trans, removing aberrant intermediates and restarting stalled biosynthesis. Knockout of this editing function leads to reduced product titers. The borrelidin biosynthetic gene cluster from Streptomyces parvulus Tu4055 contains a hitherto uncharacterized stand-alone thioesterase, borB. In this work, we demonstrate that purified BorB cleaves acyl substrates with a preference for propionate, which supports the hypothesis that it is also an editing thioesterase. The crystal structure of BorB shows a wedgelike hydrophobic substrate binding crevice that limits substrate length. To investigate the structure-function relationship, we made chimeric BorB variants using loop regions from characterized homologues with different specificities. BorB chimeras slightly reduced activity, arguing that the modified region is a not major determinant of substrate preference. The structure-function relationships described here contribute to the process of elimination for understanding thioesterase specificity and, ultimately, engineering and applying trans-acting thioesterases in biosynthetic assembly lines.

Item Type:	Journal Article
Creators:	Creators Email ORCID ORCID Put Code Curran, Samuel C. UNSPECIFIED UNSPECIFIED UNSPECIFIED Pereira, Jose H. UNSPECIFIED UNSPECIFIED UNSPECIFIED Baluyot, Marian-Joy UNSPECIFIED UNSPECIFIED UNSPECIFIED Lake, Julie UNSPECIFIED UNSPECIFIED UNSPECIFIED Puetz, Hendrik UNSPECIFIED UNSPECIFIED UNSPECIFIED Rosenburg, Daniel J. UNSPECIFIED UNSPECIFIED UNSPECIFIED Adams, Paul UNSPECIFIED UNSPECIFIED UNSPECIFIED Keasling, Jay D. UNSPECIFIED UNSPECIFIED UNSPECIFIED
URN:	urn:nbn:de:hbz:38-336289
DOI:	10.1021/acs.biochem.0c00126
Journal or Publication Title:	Biochemistry
Volume:	59
Number:	16
Page Range:	S. 1630 - 1640
Date:	2020
Publisher:	AMER CHEMICAL SOC
Place of Publication:	WASHINGTON
ISSN:	0006-2960
Language:	English
Faculty:	Unspecified
Divisions:	Unspecified
Subjects:	no entry
Uncontrolled Keywords:	Keywords Language POLYKETIDE SYNTHASE; SUBSTRATE-SPECIFICITY; MECHANISTIC INSIGHTS; CRYSTAL-STRUCTURE; PROTEIN; DOMAIN; SELECTIVITY; CYCLIZATION; SURFACTIN; REMOVAL Multiple languages Biochemistry & Molecular Biology Multiple languages
URI:	http://kups.ub.uni-koeln.de/id/eprint/33628